Retinoic acid (RA), the active metabolite of vitamin A, is an important regulator of cell proliferation, embryonic/fetal development, and adult homeostasis. Mice that are deficient for Raldh2, an enzyme critically involved in the synthesis of RA from retinaldehyde, indicate that RA is necessary for several events in heart development. These include atrial outgrowth, ventricular trabeculation and proliferation, and left-right looping morphogenesis. The aims of this study are to distinguish which of these RA function(s) are intrinsic or extrinsic to the heart lineage. RA function(s) during later stages of heart differentiation (or postnatal life) cannot be studied in these mutants. A combination of conditional knockout and transgenic overexpression strategies will be used to alter RA levels specifically in the cardiac/epicardial lineage. In combination, these models will allow to determine which of the events controlled by RA are intrinsic ("cell-autonomous") or extrinsic to the heart tube proper. The inducible transgenic mutants that will be created will also be useful to examine later role(s) of RA in ventricular differentiation. Finally, a microarray approach has been employed revealing novel targets of RA-dependent heart development and possible signal transduction pathways. These potential targets will be examined in each genetic mutant. Since variations in vitamin A levels contribute to human congenital heart malformations, the information gained in these studies may allow us to design better prenatal strategies to prevent such malformations. Moreover, since retinoids are potent regulators of cell proliferation, understanding downstream signaling could open up new strategies to reinitiate the regenerative capacity of the heart postnatally. The following questions will be addressed in this proposal: 1) Is RA acting directly on cardiac cells (i.e. in a cell-autonomous manner) to regulate atrial and sinus venosus outgrowth programs? 2) Do alterations of RA levels have latter consequences for ventricular development? 3) What molecular pathways regulate RA-dependent ventricular proliferation?